Introduction

Focal symptomatic articular cartilage defects greater than 1.5 cm in diameter, if left untreated, may progress to osteoarthritis, which represents the progressive decline of the articular surfaces of the weightbearing joints. Cartilage injuries are common, affecting around 60% of people. Their incidence is increasing and this emphasises the need for the early detection of cartilage defects and the development of new repair and regeneration strategies. No surgical technique has shown conclusive superiority among others. Chondrocytes have a limited potential for replication or migration to a site of injury. Therefore, since joint preservation and regeneration are a worldwide major goal for orthopaedic surgeons, new techniques are being proposed. These new procedures are based upon autologous tissues and mesenchymal stem cells withdrawn from bone marrow or adipose tissue, with the goal that these cells will be able to diffentiate and replicate the anatomic architecture of the osteochondral unit and inherently withstand or otherwise restore resiliency of the cartilage to biomechanical forces, while promoting anti-nflammatory effects across the articular environment, through their paracrine action, thus, finally, preventing morbidity and maintaining mobility of the aging population. Adipose tissue serves as a reservoir for MSCs. Adipose-derived MSCs can be recovered from liposuction aspirates, but their collection from human infrapatellar fat pads has also been reported. These cells have been proved to have the the potential for differentiation into cartilage, as well as bone, tendon, and muscle. The incorporation of autologous ADMSCs into scaffolds with surgical implantation has shown to be successful in the repair of full-thickness chondral defects, while the direct injection of ADMSCs has demonstrated improvement in clinical pain and function outcomes without major adverse events. Goal of our study was to assess the efficacy of the LIPO-AMIC technique (autologous matrix-induced chondrogenesis + ADSCs and adipose tissue transfer) consisting of osteochondral lesion accurate debridement, microfracturing and filling of the defect with bilayer cell-free collagen scaffold soaked in adipose regenerative product.

Content

Eighteen patients (age range: 28-58) were initially treated for symptomatic full thickness knee chondral defects using the LIPO-AMIC technique and followed prospectively for five years. After diagnostic knee arthroscopy, the first step is, under local anesthesia, the extraction, using the simple liposuction method, of 50-80 ml of lipoaspirate from the adipose tissue of the abdomen, using a dedicated single-use kit available on the market for suction and subsequent processing (filtration and micro-fragmentation) and adipose tissue grafting (Process Kit Lipogems, Lipogems International SpA, Milan, Italy), for which use we have followed the instructions provided by the manufacturer. This device progressively reduces the size of the adipose tissue clusters, while at the same time eliminating the blood residues and the oily substances, with pro-inflammatory capacity, minimizing, at the same time, thanks to the completion of the whole process just within saline solution, the risks of damaging the mesenchymal cells. The fragmentation of adipose tissue consists in different steps that make it possible to obtain purified adipose clusters product ready for the desired clinical purpose.

The second step in the surgical procedure is the repair of the focal chondral or osteochondral defect. Once identified, the chondral or osteochondral defect is carefully cleaned up to obtain stable, clean and perpendicular margins of healthy cartilage and then accurately measured. Then, a perfect copy of the defect is obtained using an aluminium model, from which the exact imprint of the defect is cut out. After carefully performing the microfractures of the subchondral bed of the defect, and having exactly cut the membrane (Chondro-Gide®, Geistlich Pharma AG, Wolhusen, Switzerland) on the basis of the model, the collagen matrix, which was immersed for a few minutes in the microframmented adipose tissue and in the stromal vascular fraction extracted from the adipose tissue of the abdomen, is inserted in articulation to directly cover the defect. The remaining part of the cells obtained from the adipose tissue is injected onto the lesion site and then sealed with fibrin glue (Tissucol, Baxter, Rome, Italy). All patients were clinically evaluated through IKDC, KOOS and VAS scores, with follow-up between 12 and 60 months. MRI examinations were performed at 6, 12 months and yearly thereafter. ADSCs have been isolated and characterized in terms of viability and cell composition using multicolor FACS analysis.

RESULTS

No complications during or after surgery were encountered. Patients showed relevant, immediate and durature improvement of the various scores already from the very initial follow-up. At intermediate and final follow-up all scores were significantly increased (p<0.001). MRI examination, completed by T2 mapping imaging, showed early subchondral lamina regrowth and progressive maturation of the repair tissue. Histological studies shown that stem cell population resided in a perivascular location (niche) with preserved architecture and where ADSCs coexisted with pericytes and endothelial cells. FACS analysis confirmed high viability and an increased percentage of endothelial cells.

DISCUSSION

Kramer et al., in 2006, demonstrated in an in vitro study that a collagen membrane is able to treat mesenchymal cells deriving from bone marrow after microfractures and that these cells have the ability to differentiate towards chondrogenic, adipogenic and osteogenic line. Dickhut et al. have performed in vitro studies demonstrating that a biphasic carrier of type I and III collagen, such as the Chondro-Gide® membrane, is able to promote the chondrogenesis of mesenchymal cells. Compared to a collagen-free membrane, it provides greater stability of the repairing tissue.

Mesenchymal stem cells (MSCs) have shown much promise with respect to their use in cartilage tissue engineering. MSCs can be obtained from many different tissue sources. Among these, adipose tissue-derived stem cells (ADSCs) have recently been under investigation as a source for cell therapies. Adipose tissue is very rich in capillary beds, thereby harboring one of the largest depots of MSCs. Adipose tissue can, in fact, provide an abundant source of ADSC’s and stromal vascular fraction and adipose tissue cells. ADSC’s have the advantage of being abundant, easy of harvest, with rapid expansion, high proliferation potential and their ability to better maintain their phenotype with respect to BMSCs. Preparation of ADSCs is also easier and less expensive than BMSCs, and the cultured lineage has shown a similar function to BMSCs in multi-lineage differentiation. For our purposes, the main interest in ADSC’s comes from the fact that these cells are multipotent, therefore able to differentiate into different types of cells including chondrocytes. The chondrogenic potential of human ADSCs has been clearly demonstrated over several years by different works. In 2002, following an in vitro differentiation process, Erickson et al. have shown that human ADSCs maintain the phenotype of chondrocytes and form cartilaginous tissue when implanted subcutaneously in vivo in immuno-deficient mice for up to 12 weeks. In numerous other studies ​​it has been shown that, under certain conditions, ADSCs can express the genes and proteins of different molecules that are specific to cartilage, including type II collagen and aggrecan, without expression of hypertrophic chondrocytes markers, such as type X collagen. However, under various defined conditions, ADSC’s may be induced to synthesize type I and type II collagen, suggesting that a cartilaginous phenotype is also possible. When kept in pellet culture or encapsulated in alginate beads and cultured with 10 ng / ml of TGF-β1 growth factor, ascorbate and dexamethasone, and have been shown to express a chondrocyte-like phenotype and synthesize type II collagen, aggrecan, link proteins and chondroitin sulfate in a time-dependent manner based on the analysis, immunoistochemistry, and the incorporation of mRNA radiotracers, with a significant increase in the synthesis of proteoglycans and proteins under condogenic conditions. Winter et al. concluded that the gene expression profile of ADSCs in chondrogenic conditions is similar to that of mesenchymal cells. Mesenchymal cells from adipose tissue at this time can, therefore, be considered to be able to form cartilaginous tissue.

The Chondro-Gide® is a resorbable collagen membrane, consisting of collagen types I and III. It’s particularity is due to it’s bilayer structure, with one compact and one porous side. The compact layer, which is cell occlusive, prevents cells from diffusion and also protects them from mechanical impact. The porous layer is made of collagen fibers in a loose, porous arrangement that favours cell invasion and attachment. This 3D collagen matrix stimulates the autologous cultured cells to differentiate into the chondrocyte phenotype and to produce collagen II and GAG. The advantage of adding adipose tissue transfer containing ADSC’s is the capability to activate the chondrocytes with regard to outgrowth and proliferation. Vice versa, the cartilage cells might favour the stem cells to differentiate along the chondrogenic lineage.

CONCLUSION

Repair of full-thickness cartilage injuries by LIPO-AMIC technique provides good to excellent clinical improvement, MRI defect filling and, at advanced and detailed histologic evaluation, high percentage of ADSCs and endothelial cell populations with high viability and niche preservation. Results resulted improved in respect to standard AMIC technique and comparable to MACI, but at significantly reduced costs. In conclusion, the LIPO-AMIC technique is an effective and safe single-step cell-based procedure to manage full-thickness focal chondral lesions of the knee.

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